JP2023181361A - Use of pcbp1 to treat hyperproliferative disease - Google Patents

Abstract

To provide compositions and methods of using gene therapy to decrease the cell migration and metastasis and reduce the tumor mass or burden.SOLUTION: The methods disclosed herein employ plasmids, vectors, and non-viral vectors that contain PCBP1 and/or PCBP1 mutants which inhibit the expression of cancer biomarkers and decrease the cell migration or tumor size. In some embodiments, a vector comprises a mutant PCBP1 nucleic acid sequence that encodes a mutant PCBP1 polypeptide. In some embodiments, the mutant PCBP1 polypeptide is not phosphorylated or not fully phosphorylated compared to wild type PCBP1.SELECTED DRAWING: Figure 1A

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/626,424, filed February 5, 2018, the contents of which are incorporated by reference in their entirety for all purposes. Incorporated.

The present disclosure relates to compositions and methods for inhibiting cell migration and cancer metastasis and reducing tumor burden. Specifically, the present disclosure provides methods and compositions for transduction of cancer cells to inhibit cancer cell migration and cancer development and/or metastasis. Compositions may be administered in vitro, in vivo, ex vivo, or in situ.

Description of Electronically Submitted Text Files The contents of text files electronically submitted with this specification are incorporated herein by reference in their entirety: Computer-readable format copy of the Sequence Listing (File name: IBEX-003/01WO_SeqList_ST25.txt, data recording date: February 5, 2019, file size 15 kilobytes).

Regenerating liver phosphatase 3 (PRL-3) is an oncogenic factor, and its activation is often associated with tumorigenesis and metastasis. Previous studies have shown that poly(rC) binding protein (PCBP1) may be able to reduce the expression of PRL-3 and inhibit the development of carcinogenesis.

To realize the promise of novel gene therapies for cancer, cancer cells must be engineered such that expression of biomarkers associated with cancer development and tumorigenesis and metastasis is reduced or prevented. It won't happen. This prevents the development of cancer cells and/or prevents them from metastasizing and minimizes invasion of tissues and organs throughout the body.

The present disclosure uses gene therapy to improve tumorigenesis, growth, cell migration, and the like by transfecting and/or transducing cancer cells with compositions encoding PCBP1 polypeptides, variants and/or variants thereof. and/or methods of inhibiting metastasis.

In some embodiments, the present disclosure provides methods of inhibiting, preventing, or reducing cell migration comprising introducing into a cell a vector comprising PCBP1 or a mutant PCBP1 nucleic acid sequence. In some embodiments, cell migration is metastasis.

In some embodiments, the present disclosure provides a method of treating cancer comprising introducing a vector comprising a PCBP1 or mutant PCBP1 nucleic acid sequence into a cancer cell.

In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.

In some embodiments, the cell is mammalian. In some embodiments, the mammalian cell is human. In some embodiments, the mammalian cell is an organ cell, tissue cell, or blood cell.

In some embodiments, the mammalian cell is a cancer cell. In some embodiments, the cancer cells are from melanoma, prostate cancer, pancreatic cancer, glioblastoma, retinoblastoma, or breast cancer.

In some embodiments, the mutant PCBP1 nucleic acid sequence encodes a polypeptide that is unable to be phosphorylated or completely unable to be phosphorylated compared to wild-type PCBP1. In some embodiments, the mutant PCBP1 is not phosphorylated or not completely phosphorylated by p21-activated kinase (PAK).

In some embodiments, the mutant PCBP1 nucleic acid comprises one, two, three, four, or five or more mutations compared to wild-type PCBP1 (SEQ ID NO: 1). In some embodiments, the mutant PCBP1 nucleic acid has a percent identity of at least 75% with wild-type PCBP1 (SEQ ID NO: 1). In some embodiments, the variant PCPB1 nucleic acid comprises the sequence of SEQ ID NO:3. In some embodiments, the variant PCBP1 nucleic acid comprises the sequence of SEQ ID NO:5.

In some embodiments, the vector comprises a mutant PCBP1 nucleic acid sequence encoding a mutant PCBP1 polypeptide. In some embodiments, the mutant PCBP1 polypeptide is not phosphorylated or not completely phosphorylated compared to wild-type PCBP1. In some embodiments, the mutant PCBP1 polypeptide is not phosphorylated or not completely phosphorylated by P21-activated kinase-1 (PAK1). In some embodiments, the mutant PCBP1 inhibits or prevents phosphorylated PCBP1 production.

In some embodiments, the mutant PCBP1 polypeptide comprises 1, 2, 3, 4, 5, or more mutations compared to wild-type PCBP1 (SEQ ID NO: 2). In some embodiments, the variant PCBP1 polypeptide has a percent identity of at least 75% with wild-type PCBP1 (SEQ ID NO: 2). In some embodiments, the variant PCPB1 polypeptide comprises the sequence of SEQ ID NO:4. In some embodiments, the variant PCBP1 polypeptide comprises the sequence of SEQ ID NO:6.

In some embodiments, the cell expresses multiple copies of a wild-type or mutant PCBP1 nucleic acid sequence. In some embodiments, the cell expresses multiple copies of a wild-type or mutant PCBP1 polypeptide.
In certain embodiments, for example, the following items are provided:
(Item 1)
A method of inhibiting, preventing or reducing cell migration comprising introducing into a cell a vector comprising a mutant PCBP1 nucleic acid sequence.
(Item 2)
A method of treating cancer comprising introducing a vector containing a mutant PCBP1 nucleic acid sequence into cancer cells.
(Item 3)
3. The method according to item 1 or 2, wherein the method is performed in vitro.
(Item 4)
3. A method according to item 1 or 2, wherein said method is performed ex vivo.
(Item 5)
3. A method according to item 1 or 2, wherein said method is carried out in vivo.
(Item 6)
The method according to item 1 or 2, wherein the cell is a mammal.
(Item 7)
7. The method according to item 6, wherein the mammalian cell is human.
(Item 8)
7. The method according to item 6, wherein the mammalian cell is an organ cell, tissue cell, or blood cell.
(Item 9)
7. The method according to item 6, wherein the mammalian cell is a cancer cell.
(Item 10)
The method according to item 9, wherein the cancer cells are cells derived from melanoma, prostate cancer, or breast cancer.
(Item 11)
3. The method according to item 1 or 2, wherein the mutant PCBP1 nucleic acid sequence encodes a polypeptide that is not completely phosphorylated.
(Item 12)
12. The method according to item 11, wherein the mutant PCBP1 is not completely phosphorylated by PAK.
(Item 13)
Item 1 or 2, wherein the mutant PCBP1 nucleic acid contains 1, 2, 3, 4, 5, or more mutations compared to wild-type PCBP1 (SEQ ID NO: 1). Method.
(Item 14)
3. The method of item 1 or 2, wherein the mutant PCBP1 nucleic acid has a percent identity of at least 75% to wild type PCBP1 (SEQ ID NO: 1).
(Item 15)
The method according to item 1 or 2, wherein the mutant PCPB1 nucleic acid comprises the sequence of SEQ ID NO: 3.
(Item 16)
The method according to item 1 or 2, wherein the mutant PCBP1 nucleic acid comprises the sequence of SEQ ID NO: 5.
(Item 17)
3. The method of item 1 or 2, wherein the vector comprises a mutant PCBP1 nucleic acid sequence encoding a mutant PCBP1 polypeptide.
(Item 18)
18. The method according to item 17, wherein the mutant PCBP1 polypeptide is not completely phosphorylated.
(Item 19)
19. The method according to item 18, wherein the mutant PCBP1 polypeptide is not completely phosphorylated by PAK.
(Item 20)
The method of item 17, wherein the mutant PCBP1 polypeptide comprises one, two, three, four, five, or more mutations compared to wild-type PCBP1 (SEQ ID NO: 2). . (Item 21)
18. The method of item 17, wherein said variant PCBP1 polypeptide has a percent identity of at least 75% with PCBP type 1 (SEQ ID NO: 2).
(Item 22)
18. The method of item 17, wherein the mutant PCPB1 polypeptide comprises the sequence SEQ ID NO: 4.
(Item 23)
18. The method of item 17, wherein the mutant PCBP1 polypeptide comprises the sequence SEQ ID NO: 6.
(Item 24)
3. The method of item 1 or 2, wherein said cell expresses multiple copies of said PCBP1 nucleic acid sequence.
(Item 25)
3. The method of item 1 or 2, wherein the cell expresses multiple copies of the PCBP1 polypeptide.
(Item 26)
3. The method of item 1 or 2, wherein the vector comprises a mutant PCBP1 nucleic acid sequence encoding one, two, and/or three domains of PCBP1.
(Item 27)
27. The method of item 26, wherein the one or more domains of the mutant PBCB1 are not completely phosphorylated.
(Item 28)
A method for monitoring cell migration and/or cancer development, which comprises introducing a vector containing a mutant PCBP1 nucleic acid sequence into cells.
(Item 29)
29. The method according to item 28, wherein the cell migration is metastasis.
(Item 30)
29. The method of item 28, wherein said cells are exposed to a therapeutic treatment.
(Item 31)
31. The method of item 30, wherein the therapeutic treatment is a chemotherapeutic treatment.
(Item 32)
29. The method of item 28, wherein said cancer cells are exposed to therapeutic treatment in vitro.
(Item 33)
29. The method of item 28, wherein said cancer cells are exposed to therapeutic treatment in vivo.

Representative lentiviral vectors containing wild-type or mutant PCBP1 sequences are shown. In this vector, PCBP1 is driven by a secondary expression cassette containing the EF1a promoter followed by an IRES-GFP reporter gene cassette. Representative AAV vectors containing wild-type or mutant PCBP1 sequences are shown. In this vector, expression of the PCBP1 sequence is driven by a secondary expression cassette containing the EF1a promoter followed by an IRES-GFP reporter gene cassette. This vector can contain a WPRE fragment inserted after the GFP sequence to enhance expression of the transgene(s). qRT-PCR analysis of gene expression in melanoma cells transfected with compositions of the present disclosure measuring the fold change between groups (ddct): GFP only versus GFP and wild-type PCBP1 (wPCBP1_0), respectively, showed 4.012 times higher upregulation of PCBP1, GFP only vs. GFP and PCBP1 S223L (mPCBP1_1) showed 3.97 times higher PCBP1 expression, GFP only vs. GFP and PCBP1 T60A & T127A (mPCBP1_2) showed 4.012 times higher upregulation of PCBP1. 02 times higher PCBP1 expression (*p<0.01). The housekeeping gene is GAPDH. Figure 3 shows GFP expression in melanoma cells after in vitro transfection of therapeutic gene vectors of the present disclosure. (A): GFP only, (B) wPCBP1_0-IRES-GFP, (C) mPCBP1_1-IRES-GFP, (D): mPCBP1_2-IRES-GFP. (Scale bar: 200 μm). Figure 3 shows PRL-3 protein expression by Western blot analysis after treatment with gene therapy vectors encoding different forms of the PCBP1 (wild type and mutant) gene in melanoma cells. Lane 1: GFP only, Lane 2: mPCBP1_1+GFP, Lane 3: wPCBP1_0+GFP, Lane 4: PCBP1_2+GFP. Beta-actin is a control. To demonstrate migration of transfected melanoma cells, a comparison of the net cell number difference in the circular detection zone (CDZ) as a percentage of migrated cells compared to all cells in the well is shown. The percentage of migrated cells (per well) in the GFP group (11.33) was significantly higher than wPCBP1_0 (wild type PCBP1, also referred to herein as "PCBP1"), mPCBP1_1, and mPCBP1_2. Of these three groups, mPCBP1_2 has the lowest percentage of migrated cells per well (1.82). (*P<0.05). Cell migration assay is shown. Cell migration assays were read on days 1 and 5 and observed under a fluorescence microscope (green filter). The top row shows the cell migration of wPCBP1_0 from days 1 and 5, the middle row shows mPCBP1_2 and mPCBP1_1, and the bottom row shows the GFP group. Column A is the cell migration well on day 1 (the white dotted circle is called the circular detection zone, or CDZ), Column B is the cell migration well on day 5 (the CDZ is the white dotted line), Column C is an expanded CDZ of column B. Net cell number differences in the circular detection zone (CDZ) migration assay are shown as a comparison of net cell number differences in the CDZ from day 1 to day 5. Melanoma cells were transfected with GFP, wPCBP1_0, mPCBP1_1, or mPCBP1_2. Figure 3 shows the fold change in PCBP1 mRNA levels in a breast cancer (BC) cell line (MCF-7). The graph shows qRT-PCR results of PCBP1 mRNA fold change in PCBP1 (WT/mutant) or GFP-only vectors transfected into MCF-7. Compared to the GFP-only group, all PCBP1+GFP groups (WT and mutant) show a significant increase in PCBP1 mRNA expression levels in breast cancer cells (fold change shown in the figure, *P<0.01). (GFP: green fluorescent protein vector. w PCBP1_0-GFP vector; mPCBP1_1 (1 site mutation (c668t)); mPCBP1_2 (2 site mutation (a178g & a379g)). PRL3 protein levels in transformed breast cancer (MCF-7) cells after treatment with vectors encoding GFP alone (lane 1), wPCBP1_0 (lane 2), mPCBP1_1 (lane 3), and mPCBP1_2 (lane 4). Western blot analysis of. The comparison ratio between the net cell difference counted in the circular detection zone (CDZ) and the fluorescence intensity of total cells per well is shown. Breast cancer cell (MCF-7) migration assay is shown. Cell migration assays were read on days 1 and 5 and observed under a fluorescence microscope (green filter). (GFP: green fluorescent protein). w PCBP1_0-GFP vector; mPCBP1_1 (one-site mutation (c668t)-GFP vector); mPCBP1_2 (two-site mutation (a178g & a379g)-GFP vector). Cell migration assay for breast cancer cells (MCF-7) transfected with GFP, wPCBP1_0, mPCBP1_1, or mPCBP1_2 is shown. Breast cancer (MCF-7) dead cell assay after PCBP1 (wild type/mutant) or GFP (control) transfection is shown. Dead cells were determined by removing the supernatant and applying a commercial live/dead cell assay kit. A: Breast cancer cell density after GFP transfection and observation using an Akeno microscope. B: Breast cancer cell density after wild-type PCBP1 transfection and observation using an Akeno microscope. C: Breast cancer cell density after mPCBP1_1 (one-site mutation) transfection and observation using an Akeno microscope. D: Breast cancer cell density after mPCBP1_2 (two-site mutation) transfection and observation using an Akeno microscope. E: Number of dead breast cancer cells measured by fluorescence intensity measurement using a microplate reader after transfection with PCBP1 (wild type/mutant type) and/or GFP vector. (GFP: cells transfected with green fluorescent protein; wP: cells transfected with wild-type PCBP1; P1: cells transfected with mPCBP1_1 (one-site mutation); P2: mPCBP1_2 (two-site mutation) cells transfected by. Same as above. Same as above. Same as above. Same as above. Figure 3 shows PCBP1 expression in pancreatic cancer cells after transfection with one of the compositions of the disclosure. The graph shows qRT-PCR results of PCBP1 mRNA fold change after vectors containing wild-type PCBP1 (treatment) or GFP (cancer cells) were transfected into pancreatic cancer cells. Compared to the GFP group, the PCBP1 group shows a significant increase in PCBP1 mRNA levels in pancreatic cancer cells (fold change shown in Figure 9A). The GFP group also showed higher PRL3 mRNA expression compared to the PCBP1 group (fold change shown in Figure 9B). Same as above. Figure 3 shows fold change in PCBP1 mRNA expression levels in treatment groups with wild type and mutant forms of PCBP1 in prostate cancer cell line (DU-145) compared to GFP group (control). The graph shows qRT-PCR results showing the fold change in PCBP1 mRNA expression in cancer cells (DU-145) transfected with vectors containing PCBP1 (WT/mutant) + GFP or GFP only. Compared to the GFP group, all PCBP1 groups (WT and mutant) showed a significant increase in PCBP1 mRNA levels in prostate cancer cells (fold change shown in the figure, *P<0.01). (GFP: green fluorescent protein vector. wPCBP1_0-GFP vector; mPCBP1_1 (1 site mutation); mPCBP1_2 (2 site mutation). Shown is the measurement of PRL3 mRNA expression levels in a glioblastoma cell line versus a cortical neuron cell line (HCN-2) by qRT-PCR. Compared to normal brain cortical cell lines, PRL3 mRNA is overexpressed in all six glioblastoma cell lines tested. A schematic diagram of an AAV vector expressing PCBP1 (wild type or mutant) under the control of a cell-specific promoter is shown. Here, expression of the PCBP1 sequence (wt, mPCBP1_1, or mPCBP1_2) is driven by a cell-specific promoter, such as the glial fibrillary acidic protein (GFAP) promoter, to treat glioblastoma in the brain. GFP or LacZ is a reporter gene used to illustrate co-localization of therapeutic proteins (eg, PCBP1) and marker protein expression in specific cell types. In vitro detection of transgene expression driven by specific promoters in glioblastoma (brain) cells. LN229 glioblastoma cells transfected with phGFAP-LacZ vector were analyzed via immunohistochemistry. Cancer cells (LN299) were observed using an Akeno microscope (panel A). Expressed protein from the LacZ gene driven by a specific promoter was detected by anti-β-galactosidase antibody A11132 (diluted 1:700). (Panel B). Northernnights™ 557 conjugated anti-rabbit antibody (NL004, R&D system) was used as the secondary antibody (dilution 1:200), resulting in a red fluorescent signal. The magnification is 20x. Same as above. In vitro detection of transgene expression driven by specific promoters in glioblastoma (brain) cells. U87 glioblastoma cells transfected with phGFAP-LacZ vector were analyzed via immunohistochemistry. Cancer cells (U87) were observed using an Akeno microscope (panel A). Expressed protein from the LacZ gene driven by a specific promoter was detected by anti-β-galactosidase antibody A11132 (diluted 1:700). (Panel B). Northernnights™ 557 conjugated anti-rabbit antibody (NL004, R&D system) was used as the secondary antibody (dilution 1:200), resulting in a red fluorescent signal. The magnification is 20x. Same as above. Figure 2 shows in vitro detection of transgene expression driven by specific promoters in kidney cells. 293FT kidney cells transfected with phGFAP-LacZ vector were analyzed via immunohistochemistry. 293FT kidney cells were observed by Akeno microscopy (Panel A). Expressed protein from the LacZ gene driven by a specific promoter was not detected by anti-β-galactosidase antibody A11132 (diluted 1:700). (Panel B). Northern nights™ 557 conjugated anti-rabbit antibody (NL004, R&D system) was used as the secondary antibody (dilution 1:200), which produces a red fluorescent signal when β-galactosidase is expressed in these cells. would have brought it. The magnification is 20x. Same as above. In vitro detection of transgene expression driven by specific promoters in ocular cells. ARPE-19 eye cells transfected with phGFAP-LacZ vector were analyzed via immunohistochemistry. ARPE-19 eye cells were observed by Akeno microscopy (panel A). Expressed protein from the LacZ gene driven by a specific promoter was not detected by anti-β-galactosidase antibody A11132 (diluted 1:700). (Panel B). Northern nights™ 557 conjugated anti-rabbit antibody (NL004, R&D system) was used as the secondary antibody (dilution 1:200), which produces a red fluorescent signal when β-galactosidase is expressed in these cells. would have brought it. The magnification is 20x. Same as above. Detection of LacZ mRNA expression driven by phGFAP promoter in different cell types by qRT-PCR. LacZ mRNA expression levels in different cells (LN229, U87, ARPE-19, and 293FT) were monitored after transfecting these cells with a vector containing LacZ under the control of hGFAP. Fold change was compared with AAV-GFP vector as a control (*p<0.05). These data indicate that LacZ protein is expressed in glioblastoma (brain) cells (e.g., U87 cells and NL229 cells) under the hGFAP promoter, but not in other cell types (e.g., ARPE-19 cells and 293FT cells). Make sure that. Cell viability of glioblastoma cell lines LN229 and U87 cells after transfection with AAV vectors containing either GFP only, wild-type PCBP1 (wP), mPCBP1_1 (P1), or mPCBP1_2 (P2). Panel A shows the viability of LN229 glioblastoma cells. Panel B shows the viability of U87 glioblastoma cells. (*p<0.05 vs. GFP). Same as above. Live/dead cell signals are shown in U87 glioblastoma cells after transfection with control or PCBP-1 containing vectors. Cells were transfected with AAV-GFP, AAV-PCBP1 (wP), AAV-PCBP1_1 (P1), or AAV-PCBP1_2 (P2) and cell viability was tested after 4 days. Live cells were stained with calcein AM (panel A) and dead cells with EthD-1 (panel B). Dead cells were significantly increased after transfection with the PCBP1-containing vector compared to cells transfected with the GFP control vector. *p<0.05 Same as above. Live and dead U87 after transfection with AAV-GFP (panel A), AAV-PCBP1 (panel B (wP)), AAV-PCBP1_1 (panel C (Mu1)), or AAV-PCBP1_2 (panel D (Mu2)) Showing staining of glioblastoma cells. Same as above. Same as above. Same as above. Shows PRL3 expression levels in U87 glioblastoma cells transfected with either AAV-GFP (lane 1), AAV-PCBP1 (lane 2), AAV-PCBP1_1 (lane 3), or AAV-PCBP1_2 (lane 4). . Western blot was used to test PRL3 protein levels. Beta-actin was used as an internal loading control. Live/dead cell signals in LN229 glioblastoma cells after transfection with control or PCBP-1 containing vectors are shown. Cells were transfected with AAV-GFP, AAV-PCBP1 (wP), AAV-PCBP1_1 (P1), or AAV-PCBP1_2 (P2) and cell viability was tested after 4 days. Live cells were stained with calcein AM (panel A) and dead cells with EthD-1 (panel B). Dead cells were significantly increased after transfection with the PCBP1-containing vector compared to cells transfected with the GFP control vector. *p<0.05 Same as above. Live and dead LN229 after transfection with AAV-GFP (panel A), AAV-PCBP1 (panel B (wP)), AAV-PCBP1_1 (panel C (Mu1)), or AAV-PCBP1_2 (panel D (Mu2)). Showing staining of glioblastoma cells. Same as above. Same as above. Same as above. Shows PRL3 protein levels in LN229 glioblastoma cells transfected with either AAV-GFP (lane 1), AAV-PCBP1 (lane 2), AAV-PCBP1_1 (lane 3), or AAV-PCBP1_2 (lane 4). . Western blot was used to test PRL3 protein levels. Beta-actin was used as an internal loading control. Live/death analysis of glioblastoma M059J cell line after transfection with AAV-GFP and AAV-PCBP1 and mutants. wP = wild type PCBP1, P1 = mPCBP1_1, P2 = mPCBP1_2. *p<0.05 compared to GFP. Same as above. Live/death analysis of glioblastoma M059K cell line after transfection with AAV-GFP and AAV-PCBP1 and mutants. wP = wild type PCBP1, P1 = mPCBP1_1, P2 = mPCBP1_2. *p<0.05 compared to GFP. Same as above. Live/death analysis of glioblastoma U118MG cell line after transfection with AAV-GFP and AAV-PCBP1 and mutants. wP = wild type PCBP1, P1 = mPCBP1_1, P2 = mPCBP1_2. *p<0.05 compared to GFP. Same as above. Live/death analysis of glioblastoma U138MG cell line after transfection with AAV-GFP and AAV-PCBP1 and mutants. wP = wild type PCBP1, P1 = mPCBP1_1, P2 = mPCBP1_2. *p<0.05 compared to GFP. Same as above. Figure 3 shows measurement of pSTAT3 (Y705) protein levels by Western blot in U87 glioblastoma cells transfected with PCBP1. Cells were transfected with AAV-GFP (lane 1), AAV-PCBP1 (lane 2), AAV-mPCBP1_1 (lane 3), or AAV-mPCBP1_2 (lane 4). Treatment with STAT3 levels downregulated all forms of PCBP1 in glioblastoma cells, with mPCBP1_2 showing the most significant effect. Beta-actin served as an internal control. Figure 3 shows measurement of pSTAT3 (Y705) protein levels by Western blot in LN299 glioblastoma cells transfected with PCBP1. Cells were transfected with AAV-GFP (lane 1), AAV-PCBP1 (lane 2), AAV-mPCBP1_1 (lane 3), or AAV-mPCBP1_2 (lane 4). Treatment with all forms of PCBP1 downregulates STAT3 levels in glioblastoma cells, with mPCBP1_2 showing the most significant effect. (Panel A). Panel B shows replicate Western blot measurements of lanes 1 and 2 in panel A. Beta-actin served as an internal control. Cell proliferation and counts of ovarian SKOV3 cells after transfection with PCBP1 or mutants are shown. GFP = control vector, wild type PCBP = PCBP1, PCBP1 mutant-1 = mPCBP1_1, PCBP1-mutant-2 = mPCBP1_2. Panel A shows microscopic observations of cells transfected with either 0.5 μg or 2.0 μg vector per well. Panel B shows the number of cells counted by microplate reader. Same as above. Detection of PCBP1 and PRL3 mRNA expression levels by qRT-PCR in SKOV3 ovarian cells after treatment. The graph shows a comparison of fold change in mRNA levels (determined by qRT-PCR) between treatment groups and controls. PCBP1 mRNA expression levels show a significant increase in all PCBP1 treatment groups, including wild type (11.5-fold increase), mPCBP1_1 (11.3-fold increase), and mPCBP1_2 (11.4-fold increase). In contrast, PRL3 mRNA levels were decreased: wild type (1.403-fold decreased), mPCBP1_1 (1.978-fold decreased), and mPCBP1_2 (1.988-fold decreased). GFP = control vector, wild type PCBP = PCBP1, PCBP1 mutant-1 = mPCBP1_1, PCBP1-mutant-2 = mPCBP1_2. Figure 3 shows the effect of wild type and mutant PCBP1 overexpression on DU145 prostate cancer cell line proliferation. GFP = treatment group without PCBP1, wP = wild type PCBP1, P1 = mPCBP1_1, P2 = mPCBP1_2. Overexpression of PCBP1 or its mutants significantly inhibited prostate cancer cell proliferation compared to controls. Figure 3 shows measurement of PRL3 protein levels by Western blot in DU145 prostate cancer cells transfected with PCBP1. Cells were transfected with AAV-GFP (lane 1), AAV-PCBP1 (lane 2), AAV-mPCBP1_1 (lane 3), or AAV-mPCBP1_2 (lane 4). Treatment with all forms of PCBP1 downregulates PRL3 levels in prostate cancer cells, with mPCBP1_2 showing the most significant effect. Beta-actin served as an internal control. Cytotoxicity (LDH) assay for normal brain cell line HCN-2 is shown. HCN-2 cells were transfected with AAV-GFP, AAV-PCBP1 (wP), AAV-mPCBP1_1 (P1), or AAV-mPCBP1_2 (P2). There was no significant difference between lactate dehydrogenase (LDH) in the treatment group compared to the GFP control group (p>0.05), but a downward trend was observed. Cells in the positive control were treated with lysis buffer to elicit maximal LDH release (left column). Experiments were performed in triplicate. Cytotoxicity (LDH) assay for normal mammary cell line MCF10A is shown. MCF10A cells were transfected with AAV-GFP, AAV-PCBP1 (wP), AAV-mPCBP1_1 (P1), or AAV-mPCBP1_2 (P2). There was no significant difference between lactate dehydrogenase (LDH) in the treatment group compared to the GFP control group (p>0.05). (Panel A). No significant differences were observed between these four groups as tested by ANOVA (p>0.05). (Panel B). Treatment with wild-type PCBP1, mPCBP1_1, or mPCBP1_2 is not cytotoxic to this normal mammary cell line. Same as above.

PRL-3 expression has been found to be elevated in metastatic cancers, including ovarian, prostate, and gastric cancers, and overexpression of PRL-3 correlates with worse liver cancer prognosis (Bardelli et al. 2003 , Polato et al., 2005, Peng et al. 2004). Overexpression of PCBP1 has previously been shown to reduce PRL-3 expression (Wang et al., 2010). The present disclosure provides novel PCBP1 mutations and/or overexpressed PCBP1 that reduce PRL-3 to a greater extent than wild-type PCBP1 polypeptides or expression levels, thus reducing tumorigenicity and migration of cells.

Accordingly, the compositions and methods disclosed herein involve genetically modified (e.g., through transfection or transduction) with mutant or overexpressed PCBP1 to reduce or prevent cell migration and/or metastasis. Contains cells. Mutant PCBP1 sequences are incorporated into vector systems for gene therapy to directly prevent, inhibit, reduce, or retard cell migration for the treatment of diseases.

Poly(rC) binding protein 1 (PCBP1)
In some embodiments, the present disclosure provides vectors containing the transcription factor poly(rC) binding protein 1 (PCBP1, also known as hnRNP-E1 and αCP1), which inhibit tumorigenesis or Cell growth or metastasis can be delayed and/or prevented. In some embodiments, PCBP1 is mammalian PCBP1. In some embodiments, PCBP1 is human PCBP1, a variant, fragment, or variant thereof. In some embodiments, PCBP1, a variant, fragment, or variant thereof prevents or reduces phosphorylation of an expressed protein. In some embodiments, the PCBP1 is a variant PCBP1_1 or PCBP1_2 disclosed herein.

In some embodiments, PCBP1 reduces cell proliferation or migration. In some embodiments, PCBP1 decreases cell viability. In some embodiments, PCBP1 increases cell death. In some embodiments, PCBP1 affects immune cell growth and/or proliferation. In some embodiments, PCBP1 treats inflammation. In some embodiments, the inflammation is associated with cancer.

In some embodiments, PCBP1 treats cancer. In some embodiments, PCBP1 treats cancer by altering the expression and/or translation of cancer biomarkers associated with tumorigenesis or metastasis. In some embodiments, PCBP1 decreases the expression and/or translation of cancer biomarkers associated with tumorigenesis or metastasis. In some embodiments, cancer biomarkers include, but are not limited to, PRL-3, CD44 variants, E-cadherin, STAT-3, and vimentin. In some embodiments, the cancer biomarker associated with metastasis is PRL-3. In some embodiments, the PCBP1 is overexpressed wild-type PCBP1 or mutated PCBP1 that inhibits PRL-3 mRNA expression. In some embodiments, the PCBP1 is overexpressed wild-type PCBP1 or mutated PCBP1 that inhibits protein expression of PRL-3.

In some embodiments, PCBP1 reduces cell migration in genetically modified (eg, transfected or transduced) cells. In some embodiments, cell migration is metastasis.

In some embodiments, the mutant PCBP1 has no or reduced phosphorylation. In some embodiments, the mutant PCBP1 has no or reduced PAK phosphorylation. In some embodiments, the mutant PCBP1 inhibits or prevents phosphorylated PCBP1 production.

In some embodiments, PCBP1 is overexpressed wild type PCBP1. In some embodiments, the cell is transduced with one or more copies of wild-type PCBP1. In some embodiments, PCBP1 is a mutant. In some embodiments, the mutant PCBP1 is a single mutant. In some embodiments, the mutant PCBP1 is a double mutant. In some embodiments, the mutant PCBP1 is a triple mutant. In some embodiments, the mutant PCBP1 polypeptide comprises the S223L, T60A, and/or T127A mutations, or a combination thereof. In some embodiments, the single PCBP1 variant polypeptide is the S223L variant (referred to herein as mPCBP1_1). In some embodiments, the dual PCBP1 mutant polypeptide is a T60A & T127A mutant (referred to herein as mPCBP1_2).

In some embodiments, PCBP1 is a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is cDNA. In some embodiments, PCBP1 contains the nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. In some embodiments, PCBP1 is the full length nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. In some embodiments, PCBP1 is a fragment of the nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. In some embodiments, PCBP1 is a variant of the nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. In some embodiments, the mutant PCBP1 nucleic acid comprises a c668t, a178g, and/or a379g mutation, or a combination thereof. In some embodiments, the single PCBP1 mutant nucleic acid is the c668g mutant (referred to herein as mPCBP1_1). In some embodiments, the double PCBP1 mutant nucleic acid is an a178g & a379g mutant (referred to herein as mPCBP1_2).

Without wishing to be bound by theory, each of the KH domains within PCBP1 has been shown to bind to the mRNA of a different protein, and the use of more than one KH domain can inhibit the translation of a given protein. Can be selectively inhibited. In some embodiments, the PCBP1 nucleic acid encodes all three K homology (KH) domains. In some embodiments, the PCPB1 nucleic acid encodes two KH domains. In some embodiments, the PCPB1 nucleic acid encodes one KH domain.

Furthermore, mutations in nuclear localization signals can alter the ability of PCBP1 to translocate into the nucleus, thus affecting subsequent gene expression. In some embodiments, PCBP1 comprises mutations in one or both nuclear localization signals. In some embodiments, alteration of one or both nuclear localization signals inhibits the ability of PCBP1 to translocate into the nucleus.

Phosphorylation also plays a role in the activity of PCBP1 (eg, unphosphorylated PCBP1 may lack activity or may exhibit greater activity). In some embodiments, the PCBP1 nucleotide sequence includes mutation(s) that affect the ability of the PCBP1 polypeptide to be phosphorylated. In some embodiments, the PCBP1 nucleotide sequence variation(s) prevents PCBP1 polypeptide phosphorylation. In some embodiments, unphosphorylated PCBP1 or a PCBP1 polypeptide that is not fully phosphorylated is not active at wild-type levels.

In some embodiments, the PCBP1 polypeptide is expressed from a polycistronic mRNA transcript. In some embodiments, the PCBP1 polypeptide is expressed from a bicistronic mRNA transcript. In some embodiments, the PCBP1 polypeptide is expressed as a fusion protein.

In some embodiments, the PCBP1 nucleotide sequence comprises point mutation(s) compared to wild-type PCBP1 (SEQ ID NO: 1). In some embodiments, the PCPB1 nucleotide sequence includes point mutation(s) that affect phosphorylation. In some embodiments, the PCBP1 point mutation increases phosphorylation. In some embodiments, the PCBP1 point mutation reduces phosphorylation. In some embodiments, the PCBP1 nucleotide sequence includes mutation(s) that can affect nuclear membrane translocation. In some embodiments, the PCBP1 nucleotide sequence comprises the point mutation(s) disclosed in Table 1.

In some embodiments, the point mutations are G13A, G128C, A178G, T299C, T299A, G326A, A379G, G527A, C652T, C688T, G676A, G700A, G781T, T808G, G814T, A871G, C947T, G1033C, C1034 T, G1048C , and/or A127G, or combinations thereof. In some embodiments, the point mutation that increases phosphorylation is G13A, A178G, T299C, T299A, G326A, A379G, G527A, C652T, G781T, G814T, C947T, G1033C, C1034T, G1048C, and/or G1067T, or Selected from the group including, but not limited to, combinations thereof. In some embodiments, the point mutation that reduces phosphorylation is selected from the group including, but not limited to, C688T, A127G, or G128C, A178G, and/or A379G, or combinations thereof. In some embodiments, the point mutation is C668T, A178G and/or A379G, or a combination thereof.

In some embodiments, the present disclosure provides mimetics, analogs, derivatives, variants, or mutants of PCBP1 (SEQ ID NO: 1). In some embodiments, the mimetic, analog, derivative, variant, or variant comprises one or more nucleic acid substitutions compared to the nucleic acid sequence of native PCBP1. In some embodiments, 1-20 nucleic acids are replaced. In some embodiments, the mimetic, analog, derivative, variant, or variant has about 1, about 2, about 3, about 4 , about 5, about 6, about 7, about 8, about 9, or about 10 nucleic acid substitutions. In some embodiments, the mimetic, analog, derivative, variant, or mutant comprises one or more nucleic acid deletions compared to the native PCBP1 nucleic acid sequence (eg, SEQ ID NO: 1). In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO: 3 (mPCBP1_1(c668t)). In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO: 5 (mPCBP1_2(a178g & a379g)).

In some embodiments, 1-20 nucleic acid residues are deleted compared to the native PCBP1 nucleic acid sequence (eg, SEQ ID NO: 1). In some embodiments, the mimetic, analog, derivative, variant, or variant has about 1, about 2, about 3, about 4 , having a deletion of about 5, about 6, about 7, about 8, about 9, or about 10 nucleic acid residues. In some embodiments, 1-10 nucleic acid residues are deleted at either end compared to the native PCBP1 nucleic acid sequence (eg, SEQ ID NO: 1). In some embodiments, 1-10 nucleic acid residues are deleted from both ends compared to the native PCBP1 nucleic acid sequence. In some embodiments, the nucleic acid sequence of the mimetic, analog, derivative, variant, or mutant is at least about 70% identical to the nucleic acid sequence of native PCBP1. In some embodiments, the mimetic, analog, derivative, variant, or mutant nucleic acid sequence is about 70%, about 80%, about 85%, About 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical. Percent identity can be calculated using the alignment program EMBOSS Needle.

In some embodiments, PCBP1 is a polypeptide. In some embodiments, PCBP1 comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, PCBP1 is the full length amino acid sequence of SEQ ID NO:2. In some embodiments, PCBP1 is a fragment of the amino acid sequence of SEQ ID NO:2. In some embodiments, PCB1 is a variant of the amino acid sequence of SEQ ID NO:2. In some embodiments, the PCBP1 variant comprises the amino acid sequence of SEQ ID NO: 4 (mPCBP1_1(S223L)). In some embodiments, the PCBP1 variant comprises the amino acid sequence of SEQ ID NO: 6 (mPCBP1_2T60A & T127A). In some embodiments, PCBP1 is a functional fragment or variant of the amino acid sequence of SEQ ID NO:2. In some embodiments, the PCBP1 polypeptide includes all three K homology (KH) domains. In some embodiments, the PCPB1 polypeptide includes two KH domains. In some embodiments, the PCPB1 polypeptide includes one KH domain.

In some embodiments, the PCBP1 polypeptide comprises one mutant K homology (KH) domain. In some embodiments, the PCBP1 polypeptide comprises two mutant K homology (KH) domains. In some embodiments, the PCBP1 polypeptide comprises three mutant K homology (KH) domains. In some embodiments, mutations in one or more KH domains affect RNA binding.

In some embodiments, the PCBP1 polypeptide sequence includes amino acid mutations relative to wild-type PCBP1 (SEQ ID NO: 2). In some embodiments, the PCPB1 polypeptide sequence includes amino acid mutations that affect phosphorylation. In some embodiments, the amino acid mutations are V5M, S43A, T60A, L100P, L100Q, C109Y, A127G, G128C, T237A, G176E, P218S, G226R, D234N, D261Y, Y270D, A272S, I291V, A316V, A345P, A34 5V , E350Q, S356I, or combinations thereof. In some embodiments, mutations that increase phosphorylation include V5M, L100P, L100Q, C109Y, G176E, D261Y, A272S, A316V, A345P, A345V, and/or E350Q, or combinations thereof. selected from the group including but not limited to. In some embodiments, mutations that reduce phosphorylation are selected from the group including, but not limited to, S43A, T60A, T127A, and/or S223L, or combinations thereof. In some embodiments, the amino acid mutation is T60A, S223L, S43A, and/or T127A.

In some embodiments, the present disclosure provides mimetics, analogs, derivatives, variants, or mutants of PCBP1 (SEQ ID NO: 2). In some embodiments, the mimetic, analog, derivative, variant, or mutant comprises one or more amino acid substitutions compared to the amino acid sequence of native PCBP1. In some embodiments, more than 20 amino acids are substituted. In some embodiments, 1-20 amino acids are substituted. In some embodiments, the mimetic, analog, derivative, variant, or variant has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, or more amino acid substitutions. In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO:4. In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO:6.

In some embodiments, the mimetic, analog, derivative, variant, or variant comprises one or more amino acid deletions compared to the amino acid sequence of the native peptide therapeutic. In some embodiments, more than 20 amino acids are deleted. In some embodiments, 1-20 amino acids are deleted compared to the amino acid sequence of the native protein agent. In some embodiments, the mimetic, analog, derivative, variant, or variant has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10, or more amino acid deletions. In some embodiments, 1-10 amino acids are deleted at either terminus compared to the amino acid sequence of native PCBP1 (SEQ ID NO: 2). In some embodiments, 1-10 amino acids are deleted from both termini as compared to the amino acid sequence of native PCBP1 (SEQ ID NO: 2). In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is at least about 70% identical to the amino acid sequence of native PCBP1 (SEQ ID NO: 2). In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, about 85%, about 90% different from the amino acid sequence of native PCBP1 (SEQ ID NO: 2). %, about 95%, about 96%, about 97%, about 98%, or about 99% identical. In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, about 85%, about 90% different from the amino acid sequence of native PCBP1 (SEQ ID NO: 2). %, about 95%, about 96%, about 97%, about 98%, or about 99% identical and retains all or most of the biological activity of native PCBP1. In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, about 85%, about 90% different from the amino acid sequence of native PCBP1 (SEQ ID NO: 2). %, about 95%, about 96%, about 97%, about 98%, or about 99% identical and has reduced or altered biological activity compared to native PCBP1.

In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, About 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical. In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, is about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical and retains all or most of the biological activity of native PCBP1. In some embodiments, the amino acid sequence of the mimetic, analog, derivative, variant, or variant is about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical and has reduced or altered biological activity compared to native PCBP1. Percent identity can be calculated using the alignment program EMBOSS Needle. The following default parameters may be used for pairwise alignment: protein weight matrix = BLOSUM62, gap open = 10, gap extension = 0.1.

Vectors and Plasmids Efficient delivery of therapeutic genes to target tissues or cells is the greatest hurdle to successful gene therapy. Because naked DNA is rapidly removed or degraded in vivo by phagocytic immune cells or extracellular nucleases, means to protect the transgene may be desired. Furthermore, due to the inefficiency of spontaneous DNA uptake, a vehicle is also required to effect tissue or cell transfer. Therefore, the DNA is usually combined with some type of gene delivery vehicle, commonly called a vector, to protect and mediate effective tissue or cell transfer of the gene of interest.

Gene delivery systems can be classified as non-biological (eg, chemical and physical approaches to introducing plasmid DNA into mammalian cells) or biological (eg, viruses and bacteria). Non-viral gene delivery systems usually involve the transferred gene being carried on plasmid DNA. The plasmids used generally do not replicate in mammalian cells.

Most commonly, recombinant viruses or naked DNA or DNA complexes are used. For example, viruses can be modified in the laboratory to provide vectors that carry modified therapeutic DNA into cells, where the virus is modified to alter the genome to alter aberrant gene expression and treat genetic diseases. can be incorporated. Alternatively, the vector may remain extrachromosomal and be expressed transiently.

In some embodiments, the present disclosure provides methods of gene therapy using viral vectors. Viruses that can be used in gene therapy include, but are not limited to, lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, replication competent vectors, vaccinia virus, and herpes simplex virus. Viral vectors that can be used in gene therapy include, but are not limited to, lentiviruses, retroviruses, adenoviruses, adeno-associated virus vectors (AAV), replication competent vectors, vaccinia virus vectors, and herpes simplex virus vectors. .

In some embodiments, the present disclosure provides methods of gene therapy using non-viral vectors. In some embodiments, the present disclosure provides methods of gene therapy using bacterial vectors. In some embodiments, methods of gene therapy involve injection of naked nucleic acid (eg, DNA or RNA). This may be performed using any suitable means known in the art. In some embodiments, the present disclosure provides methods of gene therapy using bacterial delivery systems. In some embodiments, the bacterial cell is alive, attenuated, or killed. In some embodiments, bacterial delivery systems utilize the ability of cells to attach to mammalian cells or secretion systems to deliver nucleic acids and/or proteins to target mammalian cells. In some embodiments, the bacterial delivery system is Salmonella typhi, Bifidobacterium sp., Salmonella choleraesuis, Vibrio cholera, Listeria monocytogenes, Escherichia col i, Streptococcus pyogenes, and Serratia marcescens, but are not limited thereto.

In some embodiments, the present disclosure provides methods of gene therapy using non-viral and non-bacterial vectors. In some embodiments, non-viral vectors and non-bacterial vectors are eukaryotic vectors. In some embodiments, the eukaryotic vector comprises a transposon system, a CRISPR system, a zinc finger nuclease system, or a TALEN (transcription activator-like effector nuclease). In some embodiments, the transposon system is selected from, but not limited to, Sleeping Beauty or piggyBac or a transposon system. In some embodiments, other methods for nucleic acid delivery may be used, such as arginine-rich peptides or sonoporation.

In some embodiments, the present disclosure provides methods of gene therapy using nanoparticles. In some embodiments, the nanoparticles are lipid-based nanoparticles. In some embodiments, the lipid-based nanoparticles are solid lipid nanoparticles (SLNs). In some embodiments, the lipid-based nanoparticle is a non-structured lipid carrier (NLC). In some embodiments, the nanoparticles are polymer-based nanoparticles. In some embodiments, the polymer-based nanoparticles are nanospheres or nanocapsules.

Plasmid DNA-based vectors are commonly used in gene therapy and can accommodate large segments of DNA, allowing manipulation of various regulatory elements that affect gene transfer and expression. Most basically, an expression plasmid contains an expression cassette and a backbone. An expression cassette is a transcription unit containing the gene(s) of interest and any regulatory sequences necessary for expression in the target cell. The scaffold may contain a selectable marker (eg, an antibiotic resistance gene or an auxotrophic selection gene) and an origin of replication necessary for production of the plasmid in bacteria.

Any suitable plasmid can be used in the methods disclosed herein. In some embodiments, the plasmid is a lentiviral vector. In some embodiments, the plasmid is an adeno-associated virus (AAV) plasmid. In some embodiments, representative plasmids are disclosed in FIGS. 1A-B. Any suitable promoter can be used to drive expression of the genes in the expression cassette.

In some embodiments, the plasmid includes a retroviral promoter. In some embodiments, the plasmid includes a viral promoter. In some embodiments, the plasmid includes a mammalian promoter. In some embodiments, the mammalian promoter is a human promoter. In some embodiments, the promoter is tissue or cell specific. In some embodiments, the tissue-specific promoter is selected from the human transducing α-subunit promoter, GANT2, VMD2, human IRBP, K14, IRS2, and glial fibrillary acidic protein (GFAP) promoter. Selected promoters can be used as chimeric promoters (eg, with the interphotoreceptor retinoid binding protein (IRBP) promoter). In some embodiments, tissue- or cell-specific promoters are expressed in pancreatic cells, pancreatic beta cells, skin cells, corneocytes, photoreceptor cells, epithelial cells, endothelial cells, and/or cancer cells (e.g., breast cancer cells, specific for prostate cancer cells, leukemia cells, lymphoma cells, nerve cancer cells, glioblastoma, etc.). In some embodiments, the promoter is specific for retinal or ocular tissues or cells. In some embodiments, the promoter is specific for hematopoietic cells. In some embodiments, the promoter is specific for liver tissue or cells. In some embodiments, the promoter is lung tissue or cell specific. In some embodiments, the promoter is muscle tissue or cell specific. In some embodiments, the promoter is specific for HIV-infected cells.

In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is inducible by any suitable composition or stimulus including, but not limited to, doxycycline, tetracycline, IPTG, ecdysone, or rapamycin. In some embodiments, the inducible promoter is selected from any known to those of skill in the art, including, but not limited to, TRE3G, tetracycline, Lac, ecdysone, and rapamycin promoters. In some embodiments, the promoter is constitutive. In some embodiments, the promoter is a synthetic promoter or includes an enhancer element. In some embodiments, the promoter is a hybrid promoter. In some embodiments, the hybrid promoter includes the regulatory region of the gene.

In some embodiments, the promoter is Efla, CAG, sv40, CMV, RSV, Oct4, Rex1, Nanog, GANT2, VMD2, hIRBP, TET promoter, CAAT box, GC box, GT-1 motif, I-box, AT-rich sequences, RBCS1, TRE3G, GAL1, Lap267, rapamycin, CD11a, CD11b, CD18, beta-globin promoter/LCR, immunoglobulin promoter, PEPCK promoter, albumin promoter, hAAT, SPC, SP-A, MCK, VLC1 , HIV-LTR, Tat/Rev responsive element, Tat inducible element, and FMR1.

In some embodiments, the plasmid contains all of the genes introduced via gene therapy. In some embodiments, the plasmid comprises PCBP1. In some embodiments, PCBP1 is a mutant PCBP1. In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO:3. In some embodiments, the mimetic, analog, derivative, variant, or variant comprises SEQ ID NO:5. In some embodiments, the PCBP1 mimetic, analog, derivative, variant, or variant encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

In some embodiments, introduction of a vector of the present disclosure increases gene copy number within a cell. In some embodiments, introduction of a vector of the present disclosure increases gene expression within a cell. In some embodiments, introduction of a vector of the present disclosure increases polypeptide expression within a cell. In some embodiments, the gene is PCBP1 or a variant thereof.

Diseases and Disorders The compositions and methods disclosed herein can be used on any suitable cell or tissue type. In some embodiments, the methods disclosed herein are performed in vitro. In some embodiments, the methods disclosed herein are performed ex vivo. In some embodiments, the methods disclosed herein are performed on isolated cells. In some embodiments, the methods disclosed herein are performed in cell culture. In some embodiments, isolated cells or cell culture cells are harvested from a subject and then transplanted after transfection or transduction. In some embodiments, the cell is a cancer cell.

In some embodiments, the compositions and methods disclosed herein are used in vivo. In some embodiments, the methods disclosed herein are used in situ. In some embodiments, the methods disclosed herein are directed to the eye, retina, heart, blood, white blood cells, red blood cells, platelets, vitreous humor, sclera, retina, iris, cornea, skeletal muscle, cardiac muscle, smooth muscle. , cartilage, tendons, bones, epidermis, organs, liver, heart, kidneys, lungs, stomach, gastrointestinal tract, colon, bladder, ovaries, testicles, pancreas, bone marrow, brain, neurons, and/or glands. Used to reduce cell migration in any suitable part of the body, including but not limited to.

The compositions and methods disclosed herein can be used for the treatment, prevention, amelioration, or delay of any suitable disease. For example, diseases that can be treated, prevented, ameliorated, or delayed are characterized by cell migration.

In some embodiments, the compositions and methods disclosed herein can be used to treat cancer or uncontrolled cell growth. In some embodiments, the compositions and methods disclosed herein are used to prevent, inhibit, ameliorate, or reduce metastasis or uncontrolled cell migration. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a non-solid cancer. In some embodiments, the present disclosure provides treatment for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytomas (e.g., pediatric cerebellum or cerebrum), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumors (e.g. osteosarcoma, malignant fibrous histiocytoma), brainstem glioma, brain cancer, brain tumors (e.g. glioblastoma, cerebellar star) Cytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual tract and hypothalamic glioma), breast cancer, bronchial adenoma/carcinoid, Burkitt's lymphoma, Carcinoid tumor, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibroplasia Small round cell tumors, endometrial cancer, ependymomas, esophageal cancers, Ewing sarcoma, extragonadal germ cell tumors, extrahepatic cholangiocarcinomas, eye cancers, gallbladder cancers, gastric (abdominal) cancers, gastrointestinal stromal tumors ( GIST), germ cell tumors (e.g. extracranial, extragonadal, ovarian), gestational trophoblastic tumors, gliomas (e.g. brainstem, cerebral astrocytomas, visual tract and hypothalamus), gastric carcinoids, head and neck cancers, Heart cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, hypothalamic and visual tract glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (e.g. , acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hair cell), lip and oral cavity cancer, liposarcoma, liver cancer, lung cancer (e.g., non-small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt, cutaneous T-cell Hodgkin, non-Hodgkin, central nervous system primary), medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma, oral cancer (mouth cancer), multiple endocrine tumor syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disease, myeloid leukemia, myeloid leukemia, bone marrow proliferative disorders, chronic, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, Pancreatic cancer, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma and/or germinoma, pineoblastoma and supratentorial extraneural cancer. Germinal tumor, pituitary adenoma, plasma cell tumor/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter, retinoblast cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g. Ewing family, Kaposi, soft tissue, uterine), Sézary syndrome, skin cancer (e.g. non-melanoma, melanoma, Merkel cell), small cell lung cancer, small intestine cancer, Soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric cancer, supratentorial primitive neuroectodermal tumor, T cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor , ureteral and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual tract and hypothalamic glioma, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor. Relating to, but not limited to, cancer. In some embodiments, cells transfected or transduced with compositions of the present disclosure are obtained from one or more of these types of cancer.

Administration Compositions of the present disclosure may be administered via any suitable means. In some embodiments, the route of nucleic acid administration is transdermal, injection, intramuscular, subcutaneous, oral, nasal, intravaginal, rectal, transmucosal, enteral, parenteral, topical, epidural, intracerebral, Intracerebrovascular, intraarterial, intraarticular, intradermal, intralesional, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravenous, intravesical, or intravitreal.

In some embodiments, compositions or cells transfected or transduced with compositions using the methods disclosed herein are administered to a patient. In some embodiments, compositions or cells transfected or transduced with compositions made using the methods disclosed herein are used to treat, prevent, ameliorate, or treat a disease or disorder. Administered to patients to delay onset. In some embodiments, administration of the compositions disclosed herein prevents, reduces, ameliorates, or slows cell migration in a subject. In some embodiments, administration of a composition disclosed herein prevents, reduces, ameliorates, or slows cell migration in a subject and treats, prevents, or ameliorates a disease or disorder. or delay its onset. In some embodiments, the disease or disorder is cancer or metastatic cancer. In some embodiments, administration of a composition disclosed herein reduces tumor mass/burden in a subject.

In some embodiments, the vectors, nucleic acids, or cells disclosed herein are administered once to a patient. In some embodiments, the vectors, nucleic acids, or cells disclosed herein are administered to a patient about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times. The administration may be administered twice, about 9 times, about 10 times, about 20 times, about 40 times, or more. The vectors, nucleic acids, or cells disclosed herein are administered until the symptoms of the disease or disorder ameliorate.

In some embodiments, administration of a vector or nucleic acid disclosed herein prevents, ameliorates, or reduces cell migration or proliferation in a treated patient as compared to an untreated patient or the same patient prior to treatment. cause or delay. In some embodiments, cell migration or proliferation is prevented, ameliorated, reduced, or delayed in the treated patient from day 1 to year 10. In some embodiments, administration of a plasmid or vector disclosed herein is performed on about day 1, on about day 2, as compared to cell migration or proliferation in an untreated patient or the same patient before treatment. Approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, approximately 6th week, About 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about 60th week, about 70th week, Preventing, ameliorating, reducing, or retarding cell migration or proliferation at about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of a nucleic acid or vector disclosed herein reduces cell migration or proliferation by about 1 day, about 1 week, about 1 Preventing cell migration or proliferation for months, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more to make, improve, reduce, or delay.

In some embodiments, cell migration or proliferation is about 1%, about 5%, about 10%, about 20% compared to controls or patients or in vitro cells treated with other cell migration or proliferation inhibition methods. %, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1st day, approximately 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, about 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about At 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, reduce cell migration or proliferation by about 1%; about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1. About 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, Reduce cell migration or proliferation by about 1%, about 5%, about 10% for about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more , about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

In some embodiments, administration of a vector or nucleic acid disclosed herein reduces tumor mass or burden in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, tumor mass/burden is reduced in treated patients from day 1 to year 10. In some embodiments, administration of a plasmid or vector disclosed herein is performed on about day 1, on about day 2, as compared to tumor mass or burden in an untreated patient or the same patient before treatment. Approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, approximately 6th week, About 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about 60th week, about 70th week, The tumor mass or burden is reduced at about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of a nucleic acid or vector disclosed herein provides about 1 day, about 1 week, about 1 reducing tumor mass or burden for a period of 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more do.

In some embodiments, the tumor mass or burden is about 1%, about 5%, about 10%, about 20% compared to controls or patients or in vitro cells treated with other tumor mass or burden reduction methods. %, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein is administered for about 1 day compared to controls or patients treated with other tumor mass or burden reduction methods or cells in vitro. 2nd day, 3rd day, 4th day, 5th day, 6th day, 1st week, 2nd week, 3rd week, 4th week, 5th day 6th week, 7th week, 8th week, 9th week, 10th week, 20th week, 30th week, 40th week, 50th week, 60th week At about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, the tumor mass or burden is reduced by about 1%, about 5 %, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 About 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 Reduce tumor mass or burden by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

In some embodiments, the vectors, nucleic acids, or cells disclosed herein reduce cancer biomarker expression in treated cells in vitro. In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein reduces cancer biomarker expression in treated patients. In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein reduces cancer biomarker expression in treated patients or in vitro cells from day 1 to year 10. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein is administered for about 1 day compared to controls or patients or cells in vitro treated with other cancer biomarker expression inhibition methods. 2nd day, 3rd day, 4th day, 5th day, 6th day, 1st week, 2nd week, 3rd week, 4th week, 5th day 6th week, 7th week, 8th week, 9th week, 10th week, 20th week, 30th week, 40th week, 50th week, 60th week reducing cancer biomarker expression at about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of plasmids, vectors, or cells disclosed herein is administered for about 1 day compared to controls or patients or cells in vitro treated with other cancer biomarker expression inhibition methods. , 2 days, 3 days, 4 days, 5 days, 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 The cancer biomarker expression is reduced for months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more.

In some embodiments, cancer biomarker expression is about 1%, about 5%, about 10%, about 20% compared to controls or patients or in vitro cells treated with other cancer biomarker expression inhibition methods. %, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein is administered for about 1 day compared to controls or patients or cells in vitro treated with other cancer biomarker expression inhibition methods. 2nd day, 3rd day, 4th day, 5th day, 6th day, 1st week, 2nd week, 3rd week, 4th week, 5th day 6th week, 7th week, 8th week, 9th week, 10th week, 20th week, 30th week, 40th week, 50th week, 60th week At about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, reduce cancer biomarker expression by about 1%, about 5 %, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 About 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 Reduce cancer biomarker expression by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein reduces metastasis in an organ or tissue in vitro or ex vivo. In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein reduces metastasis in a tissue, organ, or treated patient. In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein reduces metastasis in treated patients or in vitro or ex vivo organs or tissues from day 1 to year 10. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1st day, approximately 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, about 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about Metastasis is reduced at 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months , about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more.

In some embodiments, the metastasis is about 1%, about 5%, about 10%, about 20%, about 30%, about 40% compared to controls or patients treated with other metastasis inhibition methods. , about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1st day, approximately 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, about 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about At 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, about 1%, about 5% of metastases , about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week , about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or For about 10 years or more, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% , about 90%, or about 100%.

In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein increases cell death in a tissue, organ, or treated patient. In some embodiments, administration of vectors, nucleic acids, or cells disclosed herein increases cell death in the treated patient or in vitro or ex vivo organ or tissue from day 1 to year 10. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1st day, approximately 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, about 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about Metastasis is reduced at 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months , increases cell death over a period of about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more.

In some embodiments, the cell death is about 1%, about 5%, about 10%, about 20%, about 30%, about 40% compared to controls or patients treated with other cell killing methods. %, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1st day, approximately 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, about 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about At 60 weeks, about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, reduce cell death by about 1%, about 5 %, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein provides about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week , about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or Reduce cell death by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% over about 10 years or more %, about 90%, or about 100%.

In some embodiments, administration of a nucleic acid, vector, or cell disclosed herein reduces symptoms of a disease or disorder in a treated patient as compared to an untreated patient or the same patient prior to treatment. In some embodiments, symptoms of the disease or disorder are measured in the treated patient from day 1 to year 10. In some embodiments, the administration of the nucleic acids, vectors, or cells disclosed herein provides a reduction in symptoms of the disease or disorder in an untreated patient or in the same patient prior to treatment on about day 1, at 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, approximately 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about 60th week, about Reducing symptoms of a disease or disorder at 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein reduces the symptoms of the disease or disorder by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, Alleviating symptoms of a disease or disorder for about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more.

In some embodiments, the symptoms of the disease or disorder are about 1%, about 5%, about 10%, about 20%, compared to the symptoms of the disease or disorder in an untreated patient or in the same patient before treatment. reduced by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, the administration of the nucleic acids, vectors, or cells disclosed herein provides a reduction in symptoms of the disease or disorder in an untreated patient or in the same patient prior to treatment on about day 1, at 2nd day, approximately 3rd day, approximately 4th day, approximately 5th day, approximately 6th day, approximately 1st week, approximately 2nd week, approximately 3rd week, approximately 4th week, approximately 5th week, approximately 6th week, about 7th week, about 8th week, about 9th week, about 10th week, about 20th week, about 30th week, about 40th week, about 50th week, about 60th week, about At about 70 weeks, about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years, about 1%, about 5%, Reduce by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein reduces the symptoms of the disease or disorder by about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, About 1%, about 5%, about 10%, about 20% of symptoms of a disease or disorder for about 5 months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more %, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

In some embodiments, administration of a vector or nucleic acid disclosed herein reduces symptoms of cancer in a treated patient compared to an untreated patient or the same patient prior to treatment. In some embodiments, cancer symptoms are measured in treated patients from day 1 to year 10. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein is administered at about 1 day, about 2 days, compared to the symptoms of cancer in an untreated patient or in the same patient before treatment. 3rd day, 4th day, 5th day, 6th day, 1st week, 2nd week, 3rd week, 4th week, 5th day, 6th day 7th week, 8th week, 9th week, 10th week, 20th week, 30th week, 40th week, 50th week, 60th week, 70th week reducing cancer symptoms at about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein improves symptoms of cancer by about 1 day, about 2 days, About 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 The symptoms of the cancer are reduced for months, about 6 months, about 1 year, about 2 years, about 5 years, or about 10 years, or more.

In some embodiments, the symptoms of cancer are about 1%, about 5%, about 10%, about 20%, about 30%, compared to the symptoms of cancer in an untreated patient or the same patient before treatment. reduced by about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein is administered at about 1 day, about 2 days, compared to the symptoms of cancer in an untreated patient or in the same patient before treatment. 3rd day, 4th day, 5th day, 6th day, 1st week, 2nd week, 3rd week, 4th week, 5th day, 6th day 7th week, 8th week, 9th week, 10th week, 20th week, 30th week, 40th week, 50th week, 60th week, 70th week About 1%, about 5%, about 10% of cancer symptoms at about 80 weeks, about 90 weeks, about 100 weeks, about 1 year, about 2 years, or about 3 years Reduce by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, administration of the nucleic acids, vectors, or cells disclosed herein improves symptoms of cancer by about 1 day, about 2 days, About 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 Reduce symptoms of cancer by about 1%, about 5%, about 10%, about 20%, about 30% over a period of about 1%, about 5%, about 10%, about 20%, about 30% %, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

In some embodiments, the present disclosure provides methods for accurately tracking cells transfected with compositions of the present disclosure. In some embodiments, these cells are traced to determine cell behavior. In some embodiments, these cells are traced to determine their migration. In some embodiments, these cells are traced to determine their metastasis (or lack thereof). In some embodiments, these cells are traced to determine their response to any drug administered either in vitro or in vivo. In some embodiments, the cells are also exposed to a therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the cells are exposed to the therapeutic agent in vitro. In some embodiments, the cells are exposed to the therapeutic agent in vivo.

Co-administration In some embodiments, the patient is subjected to additional therapy. In some embodiments, a patient is treated with a composition of the present disclosure as part of anti-cancer therapy. In some embodiments, anti-cancer therapy is selected from, but not limited to, radiation therapy, chemotherapy, immunotherapy, hormone therapy, stem cell transplantation, and CAR-T therapy. Anti-cancer therapy may be administered before, concurrently with, or after administration of the compositions of the present disclosure.

Kits The present disclosure also provides kits for reducing, preventing, ameliorating, or slowing cell migration or metastasis. In some embodiments, the kit includes a vector or nucleic acid of the present disclosure. The kit can further include a label or printed instructions directing the use of the described reagents. The kit can further include the treatment to be tested. The kit is applied for the prevention, reduction, amelioration or delay of cell migration or metastasis in vitro and in vivo.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

As used herein, "anti-cancer gene" refers to any gene that prevents, retards, inhibits, or otherwise modifies the expression or activity of an oncogene. In some embodiments, the anti-oncogene is a tumor suppressor gene. In some embodiments, the anti-cancer gene reduces expression of cancer biomarkers associated with metastasis.

Singular forms such as "a," "an," and "the" are used throughout this application for convenience and unless context or explicit statement indicates otherwise, singular forms refer to plural forms. It should be understood that it is intended to include shapes. All numerical ranges are to be understood to include any numerical point within that numerical range, and each numerical point should be construed as individually recited. The endpoints of all ranges directed to the same component or property are intended to be inclusive and independently combinable.

As used herein, the word "comprising" and variations thereof mean that the enumeration of items in the list includes other similar It is intended to be non-limiting so as not to exclude any items. Similarly, the terms "can" and "may" and variations thereof mean that an embodiment can include or may include a particular element or feature. is intended to be non-limiting and not to exclude other embodiments of the present technology that do not include. Although the open-ended term "comprising" is used herein as a synonym for terms including, containing, or having to describe and claim the present disclosure, the present technology or embodiments thereof may alternatively be described using more restrictive terminology such as "consisting of" or "consisting essentially of" the listed ingredients.

The term "about" when used herein to refer to a quantity expressed as a number includes "exactly" plus or minus up to 10% of the referenced numerical expression. When the term "about" is used in reference to a range of values, the term "about" refers to both the minimum and maximum values of the range (e.g., "about 1 to 50 μm" refers to "about 1 μm"), and the term "about" refers to both the minimum and maximum values of the range. ~about 50 μm”). The term "intimately associated" when used herein to describe the spatial relationship between two or more components of a composition, such as in mixtures, coatings, and matrices. Refers to components that are intimately mixed.

The terms "transduction" and "transfection" are used interchangeably herein and refer to the introduction of genetic material into a cell. Any suitable genetic modification means are contemplated by this disclosure.

The present disclosure is further illustrated by the following non-limiting examples.

Example 1 - Construction of a vector expressing PCBP1 mutant polypeptide Studies have shown that PRL-3 mRNA expression is elevated in metastatic cancers, including ovarian cancer and gastric cancer, and that overexpression of PRL-3 is associated with liver cancer prognosis. It has been shown that there is a negative correlation (Bardelli et al. 2003, Polato et al., 2005, Peng et al. 2004). PCBP1 is known to regulate PRL-3 expression, and the inverse correlation between PCBP1 and PRL-3 expression suggests that PCBP1 may suppress PRL-3 mRNA translation. Downregulation of PRL-3 is accompanied by downregulation of the phosphorylated active form of Akt (pSer473) (Wang et al. 2010). For example, cancer cells that exhibit increased Akt-2 (pSer474) phosphorylation also exhibit significantly reduced PCBP1 expression (Brown et al., 2016).

As shown in Figures 1A and 1B, we generated AAV lines containing lentiviral gene therapy vectors (LVV) or/and PCBP1 genes, respectively. This vector system contains the human elongation factor-1α (EF1a) promoter and an IRES element that provides internal initiation of protein translation from the mRNA molecule. Thus, a bicistronic mRNA is created in which the IRES motif can initiate independent and distinct translation of a second gene from a single PCBPC-IRES-GFP mRNA. Based on the strategy in Table 2 below, we constructed several PCBP1 mutants designed to reduce protein phosphorylation by PAK1. PCBP1 mutant nucleotide sequences were cloned into the vectors shown in Figures 1A-B to create vector A (mPCBP1_1, S223L) and vector B (mPCBP1_2, T60A & T127A). Mutations in each PCBP1 mutant prevent PCBP1 phosphorylation by PAK1. Turning on the different genes is driven by appropriate promoter(s). PAK1 phosphorylates PCBP1 at both amino acids 60 and 127, so mutations at these sites prevent PCBP1 phosphorylation by PAK1.

Example 2 - PCBP1 mutants are expressed at higher levels than the endogenous wild type To test whether the PCBP1 S223L and PCBP1 T60A & T127A mutations affected transcription levels, PCBP1 expression in melanoma cells was Determined by qRT-PCR. Figure 2 shows the analysis of fold change between groups (ddct). Comparison of GFP expression and wild type PCBP1 expression (wPCBP1_0) shows that PCBP1 expression level was 4.012 times greater. Comparison of GFP expression and PCBP1 S223L (mPCBP1_1) expression shows that PCBP1 S223L expression level was 3.97 times greater. Comparison of GFP expression with that of the PCBP1 S223L mutant shows that the PCBP1 T60A & T127A (mPCBP1_2) expression level was 4.02 times greater. Wild type PCBP1 and the two mutant forms show similar mRNA expression. The housekeeping gene used in this assay is GAPDH.

Example 3 - Expression of PCBP1 in Melanoma Cells Melanoma cells were divided into four groups, each group containing: A) GFP only, B) wild type PCBP1 (wPCBP1_0-IRES-GFP) and GFP, 3) PCBP1 S223L and GFP (mPCBP1_1-IRES-GFP), or D) PCBP1 T60A &
Transfected in vitro with a vector containing T127A (mPCBP1_2-IRES-GFP). Expression of GFP was observed 24-48 hours after transfection of lentiviral vectors containing either wild-type or mutant PCBP1 using the Lipofectamine kit (Invitrogen Inc.) (Figure 2). . Transfection rates were approximately 90% in each case (data not shown). Variations in GFP expression levels may be due to different vectors. Without wishing to be bound by theory, PCBP1 is upstream of GFP in the vector. When PCBP1 efficiency is 100%, the downstream gene (GFP) exhibits lower expression efficiency (eg, about 60%-80%). The GFP expression efficiency is higher because the control vector contains only GFP and no PCBP1 sequence. There is no evidence that PCBP1 and GFP interact with each other.

For Western blots, cell culture dishes were placed on ice briefly after PCPB1 treatment, and cells were washed three times with ice-cold PBS. Ice-cold lysis buffer (containing protease inhibitors) was then added to the cells. Adherent cells were scraped from the dish and the cell suspension was transferred to a pre-chilled microcentrifuge tube, which was constantly agitated for 30 min at 4°C and then centrifuged. After centrifugation, the tubes were placed on ice, the supernatant was aspirated into a fresh tube, and the pellet was discarded. After protein quantification, the protein concentration of each cell lysate was determined by adding an equal volume of 2× Laemmli sample buffer. Samples were boiled in this sample buffer at 100°C for 5 minutes. Equal amounts of protein from treatment and control groups were loaded into each well of an SDS-PAGE gel along with molecular weight markers. The protein concentrations tested ranged from about 20-30 μg, or about 10-100 ng of purified protein from cell lysates or tissue homogenates. Gels were electrophoresed at 100V for 1-2 hours. The proteins on the gel were then transferred to an activated nitrocellulose membrane using transfer buffer and the Bio-Rad Semi-Transfer system (Bio-Rad, Calif.). Membranes were blocked by using blocking buffer for 1 hour at room temperature or overnight at 4°C and then washed three times (5 min/each wash) with TBST buffer before antibody staining was started. The membrane was then incubated with the chemiluminescent substrate for approximately 5 minutes to visualize the protein signal according to the kit manufacturer's recommendations (see Figures 3 and 6).

Figure 3 shows PRL-3 protein expression in cells transfected with gene therapy vectors encoding different forms of PCBP1. As shown in Figure 3, transfection of melanoma cells with a vector containing wild-type PCBP1 reduces PRL-3 expression compared to a GFP-only vector. Transfection of cells with vectors containing either of the PCBP1 mutants further reduced PRL-3 expression, with the double PCBP1 T60A & T127A mutant even more so than transfection with the single PCBP1 S223L mutant. Reduces PRL-3 expression to a large extent.

Example 4 - Transfection with mutant PCBP1 reduces melanoma cell migration The effect of mutant, dephosphorylated PCBP1 variants on cell migration was tested using a cell migration assay. Briefly, melanoma cells transfected with GFP alone, wild-type PCBP1 (wPCBP1_0-IRES-GFP) and GFP, or PCBP1 T60A & T127A (mPCBP1_2-IRES-GFP) were observed for 5 days post-transfection.

Equal numbers of cells from each treatment group or/and control were plated in 24 (or 6) wells for transfection with a vector encoding a marker protein (i.e., GFP) using the Lipofectamine kit (Invitrogen Inc). plated. The GFP-transfected cells were then suspended in medium containing trypsin (0.1%) and washed with culture medium. Equal numbers of cells (e.g., approximately 80,000 cells from each individual group) were then repopulated in a 100 μL loading volume containing the same number of cells in each well of a 96-well plate. Evenly distributed on Oris migration assay plates (Oris Cell Migration Assay kit). Each well of the dish containing Oris™ Cell Seeding Stopper (OCS) was then incubated in a humidified chamber (37°C, 5% CO2) for 5-10 hours (cell line dependent) to allow cell attachment. I made it. Before counting cells, use the Oris™ Stopper tool to remove the OCS and identify any new cells that have migrated inside this CDZ, called the "Circle Detection Zone (CDZ)", as new migrated cells. Created an area for detection. Cells expressing GFP within the CDZ were then counted at each time point using a microplate reader (Molecular Devices, LLC) or/and under a fluorescence microscope (BD Biosciences).

Cells were visualized and analyzed by fluorescence microscopy for GFP expression. Although GFP reflects the expression of the gene in each transfected cell, the intensity of GFP may decrease the level of intensity over time, as shown in the results, the fluorescence intensity is less Not lost. The total number of cells representing the intensity of each well was determined at the same time point (eg, day 0 or day 5). Because the GFP-bearing cells were transduced with the vector encoding the GFP-containing gene rather than by immunostaining with a fluorescent dye, the fluorescent signal is not stripped from the substrate. Cells that do not show a fluorescent signal were not transfected with the vectors of this disclosure and therefore are not counted and are not limited in this analysis.

The results of this cell migration assay are shown in Figures 4A-C. In FIG. 4B, the white dotted circle is the circle detection zone (CDZ). Row A is day 1, row B is the cell migration well at day 5 (CDZ is a white dotted line), and row C is the expanded CDZ from row B. Table 3 shows quantified data from cell migration assays where the double T60A & T127A mutant shows reduced net cell migration within the CDZ compared to both wild-type PCBP1 and GFP-only cells.

Figures 4A-4C show cell migration adjusted to the percentage of cells per well. The percentage of migrated cells (per well) in the GFP group (11.076) was significantly higher than wild type PCBP1 (wPCBP1_0) and PCBP1 T60A & T127A (mPCBP1_2). Among these three groups, mPCBP1_2 showed the lowest percentage of migrated cells per well (2.082). These values were calculated as follows.

At each time point, the total number of cells was detected by a microplate reader (as a reference denominator). Cells were also counted (as molecules) as migrating cells per time point within the area of the circular detection zone (CDZ). The adjusted net cell difference within the CDZ was calculated according to the formula shown below.

Example 5 - Transfection with mutant PCBP1 reduces breast cancer cell migration Breast cancer cells from the MCF-7 cell line were transfected with GFP alone, wild type PCBP1 (wPCBP1_0-IRES-GFP) and GFP, or PCBP1 T60A & T127A The cells were transfected with a vector containing (mPCBP1_2-IRES-GFP) and observed for 5 days after transfection. Migration of these cells was observed as detailed above. Table 4 shows the net cell migration within the CDZ of different transfected cells. Figures 7A-C show cell migration adjusted to the percentage of cells per well. The percentage of migrated cells (per well) in the GFP group was significantly higher than wild type PCBP1 (wPCBP1_0) and PCBP1 T60A & T127A (mPCBP1_2). Among these three groups, mPCBP1_2 showed the lowest percentage of migrated cells per well.

Example 6 - Use of PCBP1 variants to inhibit tumor metastasis Design of animal models to study cancer gene therapy:
Experimental Design: Cancer cell lines (i.e., MCF-7, breast cancer model - ATCC for BC) are tumorigenic in mice and immunodeficient animals (i.e., nude mouse line from Charles River Lab, NC: BALB). /c) can be used to study breast cancer (BC) tumor growth. These cells will be used to develop precision in vivo methods for testing therapeutic efficacy. It is first prepared for an injectable cancer cell line that is stably transduced by a vector carrying a labeled gene, ie, encoding red fluorescent protein (RFP). This allows for monitoring cancer cell growth and migration, exemplified or monitored by fluorescence bioimaging systems or fluorescence microscopy. After preparing the cancer cells, they are injected subcutaneously into mice to develop breast cancer tumors one week later. By using this new method, cancer cells will be precisely traced and studied during their metastasis after treatment with gene therapy vectors such as AAV-PCBP1m.

The procedure is carried out as follows with an example of a BC study.

(1) RFP was delivered to a BC cell line (MCF-7, ATCC), such as by using a lentiviral scaffold to integrate the RFP gene into the cell genome, for stable long-term expression of the RFP reporter gene. After confirming the successful introduction of RFP into the cells (by using fluorescence microscopy or FACS), select and inject BC cells.

(2) Divide the mice into four groups (n=6). RFP-BC cells are injected into the flank of mice under the same conditions (same number of cells, injection time) in all groups. BC tumor size will be assessed and recorded daily or twice weekly. The experimental design and expected outcomes will be as described in Table 5 below.

The vector containing the therapeutic gene is injected directly into the tumor. Mice are sacrificed for histological studies 3 weeks after treatment. Tumor tissue will be extracted for immunohistochemistry (IHC) assay of RFP, GFP, PRL-3, PCBP1, and SOX2 (BC cell markers). Detect RFP and GFP on these tissues to confirm BC cell growth and vector delivery as described in the table above. Extract additional tissues (including lung, liver, brain, and pancreas) for tumorigenicity and metastasis studies. RFP and GFP are detected within these tissues to monitor whether BCs have migrated into these tissues. qRT-PCR, WB, and IHC will also be performed. Proteomic studies are performed by two-dimensional gel electrophoresis combined with mass spectrometry techniques to compare protein expression in control versus PCBP1 mutant transduced mice. Differences between experimental groups are compared using Student's t-test. Statistical significance is determined as P<0.05.

(3) Outcome analysis for post-treatment results can be anticipated as in Table 6 below.

Example 7 - Treatment of Glioblastoma by Expressing PCBP1 in Tumor Cells Elevated levels of PRL3 mRNA are found in glioblastoma cells. As shown in Figure 11, increased PRL-3 expression in six different glioblastoma cell lines (U138MG, M059K, M059J, U118MG, U87, and LN229) compared to expression in normal brain cortical cell lines. was detected. Cell lines U87 and LN229 showed the greatest increase in PRL-3 mRNA expression and were selected for further testing.

Overexpression of wild-type or mutant PCBP1 reduces PRL3 expression in glioblastoma cells. As previously demonstrated, expression of wild-type or mutant PCBP1 reduces the expression of PRL-3, a gene associated with metastasis. decreased and inhibited cell migration. To test the effect of overexpression of the PCBP1 sequence, the present disclosure under the control of a glioblastoma cell-specific promoter, glial fibrillary acidic protein (GFAP), which allows expression of AP-PCBP1 in glioblastoma cells. Six different glioblastoma cell lines were transfected with rAAV9 expression vectors containing wild-type or mutant PCBP1 sequences (see Figure 12). Figures 13-16 demonstrate the specificity of this vector expression in glioblastoma cells. Figure 13 shows immunohistochemical analysis of LN299 glioblastoma cells treated with phGFAP-LacZ vector. Figure 14 shows immunohistochemical analysis of LN299 glioblastoma cells treated with phGFAP-LacZ vector. As shown in Figures 13B and 14B, the LacZ gene driven by the GFAP promoter was transfected with anti-β-galactosidase-specific antibody A11132 (ThermoFisher at a dilution of 1:700) in both transfected glioblastoma cell lines. Detected by. However, β-galactosidase was not detected in kidney (Figure 15) or eye (Figure 16) cells, indicating that this vector expresses its cargo only in glioblastoma cells. A comparison of LacZ mRNA expression levels is shown in FIG. 17.

Transfecting 2 micrograms of AAV vectors containing 1) GFP only, 2) wild-type PCBP1, 3) mPCBP1_1, or 4) mPCBP1_2 into LN229 and U87 glioblastoma cell lines using the Vybrant MTT kit. Cell proliferation was tested by. As shown in Figures 18A-B, overexpression of wild-type PCBP1 or either mutant significantly inhibits glioblastoma cell proliferation compared to non-treatment with AAV-GFP vector controls. Furthermore, overexpression of PCBP1 mRNA increases the number of dead cells in the culture, as shown in Figures 19 and 20. After transfection with AAV-GFP and AAV-PCBP1 and mutants, dead and live U87 glioblastoma cells were analyzed using the LIVE/DEAD™ Viability Kit (Thermofisher, Inc.). Figure 19A shows that control-treated cell cultures contain significantly more viable cells than cells transfected with vectors containing wild-type or mutant PCPB1 sequences; We show that these PCBP1 cell cultures contain significantly more dead cells than control-treated cell cultures. Figures 20A-D show staining of live/dead U87 cells after transfection with either the GFP control vector or the vector containing PCBP1 sequences. As shown in Figure 21, treatment with all forms of PCBP1 downregulated PRL3 protein expression in the U87 glioblastoma cell line, with the greatest reduction in PRL3 expression observed in cells treated with mPCBP1_2.

These results were not specific to the U87 cell line and were also demonstrated in other glioblastoma cell lines. Transfection of LN299 glioblastoma cells with wild-type or mutant PCBP1 increased the number of dead cells in the cell culture (Figures 22 and 23) and decreased the level of PRL3 protein expression (Figure 24). Again, treatment with mPCBP1_2 showed the greatest reduction in PRL3 protein expression (Figure 24). Similar effects on cell viability were observed in M059J (Figure 25), M059K (Figure 26), U118MG (Figure 27), and U138MG (Figure 28) glioblastoma cell lines.

Overexpression of wild-type or mutant PCBP1 reduces STAT3 expression in glioblastoma cells. Similar to PRL3, expression of STAT3, a cancer marker, is reduced by transfection with vectors containing wild-type or mutant PCBP1 sequences. decreased in glioblastoma cells. Figures 29 and 30 show that STAT3 protein expression levels are reduced in U87 and LN299 glioblastoma cells transfected with wild-type PCBP1, mPCBP1_1, or mPCBP1_2, with mPCBP1_2-treated cells showing the greatest reduction in STAT3 protein levels. Show that there is.

Example 8 - Treatment of Ovarian and Prostate Cancer by Expressing PCBP1 in Tumor Cells To demonstrate that the effects of PCBP1 overexpression are not limited to glioblastoma cells, the SKOV3 ovarian cancer cell line was isolated from wild-type or Transfected with a vector containing the mutant PCBP1 sequence. SKOV3 cells were transfected with either 0.5 μg or 2.0 μg of lentivector containing GFP, PCBP1, mPCBP1_1, or mPCBP1_2. Cell proliferation was assayed using MTT (Thermofisher Inc.). As shown in Figure 31, transfection of all three PCBP1-containing vectors increased inhibition of cell proliferation in a dose-dependent manner. Furthermore, FIG. 32 shows that transfection with wild-type or mutant PCBP1 sequences reduces PRL3 mRNA expression.

Similar results were observed in DU145 prostate cancer cells transfected with vectors containing wild-type or mutant PCBP1 sequences. Figure 33 shows that cells overexpressing PCBP1 showed a significant decrease in cell proliferation compared to control cells. Figure 34 shows that PRL3 protein expression levels are similarly reduced, with the mPCBP1_2 mutant showing the greatest reduction.

Example 9 - Overexpression of wild-type or mutant PCBP1 is not toxic to non-cancerous cells To demonstrate that overexpression of PCBP1 is not toxic to non-cancerous cells, wild-type or mutant Two normal cell types were transfected with vectors containing mutant PCBP1 and lactate dehydrogenase (LH) assays were performed. Lactate dehydrogenase is released into the medium from injured cells, and therefore increased levels of LH are a biomarker of cytotoxicity and cell lysis. Normal brain cells (HCN-2) and normal breast cells (MCF10A) were incubated with vectors containing 1) GFP alone (e.g., no PCBP1 treatment), 2) wild-type PCBP1, 3) mPCBP1_1, and 4) mPCBP1_2, respectively. was transfected with. Figures 33-34 show no significant killing of normal brain and breast cells by treatment with PCBP1.

INCORPORATION BY REFERENCE All cited references, applications, publications, patents, and patent publications are incorporated herein by reference in their entirety for all purposes.
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Invention described in the specification.